The importance has been noted of carrying out analysis and measurements right at the site or in the vicinity of the site where analysis and measurements are required (hereinafter referred to as “POC (point of care) analysis etc.”), such as analysis for bedside diagnosis which carry out measurements necessary for a medical diagnosis at the patient's side (POC analysis), analysis of toxic substances in rivers or wastes carried out at the sites, that is, on rivers, waste disposal centers and the like, and tests for contamination carried out at the site of cooking foods, harvesting crops, or importing foods. Then, importance has been attached to the development of detection methods and detection apparatus which are applied to such POC analysis etc. in recent years.
In the detection method and detection apparatus which are applied to such POC analysis etc., it is required that an analysis is simple, brief, and inexpensive. Furthermore, in a medical diagnosis or an environmental analysis, in order to perform comparison with a reference value, which a national government defines, in sufficient precision, it is generally required that a high sensitivity analysis is performed.
Recently, μ-TAS (micro total analysis system), which has a groove, whose depth is from tens μm to hundreds μm, on a flat glass or silicon chip at the largest 10 by 10 cm square in dimensions, or a few by few cm square or smaller in dimensions, and performing all of reactions, separation, and detection for a short time in this groove has been actively studied (for example, Japanese Patent Laid-Open No. 2-245655).
The adoption of μ-TAS has advantages that the amounts of samples, reagents for detection, and waste materials and waste fluid of consumables used for the detection are reduced, and necessary detection time is also short in general.
In addition, a method of forming a chip with resin(R. M. Mccormick et al., Anal. Chem., vol. 69, 2626–2630, 1997, Japanese Patent Laid-Open No. 2-259557, and Japanese Patent No.2639087 (registered on Apr. 25, 1997 for Shimadzu Corp.)) for developing an inexpensive disposable chip is also proposed.
However, since optical path length is dozens to hundreds μm that is 1/10 to 1/100 of that under usual conditions, in inverse proportion to it, it is required that the sensitivity of a detection apparatus is 10 to 100 times as high as that under usual conditions when optically detecting a measuring object in μ-TAS.
Up to now, a photoinduced fluorescence method or a chemiluminescence method which use a luminescence phenomenon from a measuring object and analyze the concentration and the like of the measuring object from the quantity of light of its luminescence have been adopted in a highly sensitive apparatus which optically detects the measuring object. However, the photoinduced fluorescence method has a problem that background becomes large by fluorescence from other objects than a measuring object in practical samples with many impurities since the light with wavelength near an ultraviolet ray is generally used in many cases as a light source to excite the measuring object. In addition, since a measuring object is limited to fluorescent material, it is not general as a detection method in clinical inspection such as an analysis of blood components.
Furthermore, although there is an advantage that the chemiluminescence method does not need a light source for excitation, it has not versatility similarly to the photoinduced fluorescence method.
On the other hand, as a general detection method, there is an absorption photometry which analyzes the concentration and the like of a measuring object by the absorbance of light.
Since the absorption photometry is a method applicable to any object so long as it absorbs the light with wavelength for excitation, it has been used as a detection method with very high versatility.
In addition, since the sensitivity of absorption photometry is low in comparison with the photoinduced fluorescence method or chemiluminescence method, the concentration sensitivity of the absorption photometry has been increased by providing dozens mL of measuring object, which is sufficient quantity, and making optical path length be 1 cm which is long. However, in μ-TAS, since the optical path length becomes 1/10 to 1/100 as described above, the absorption photometry has a problem that sensitivity is low when it is applied to μ-TAS although it is a common and highly versatile detection method.
As detection methods which solve the above-described problems simultaneously, photothermal spectroscopy methods are mentioned. These detection methods are methods of utilizing a phenomenon of a measuring object absorbing light that is usually a laser beam with the same wavelength as the absorption wavelength of the measuring object (hereafter, this light will be described as excitation light), and emitting heat (photothermal effect) to a surrounding solvent following relaxation process, and analyzing the concentration and the like of the measuring object by measuring the heat. The photothermal spectroscopy methods have a characteristic that the amount of absorption of light, i.e., the heat can be directly measured against the absorption photometry indirectly measuring the amount of absorption of light as the amount of decrease of transmitted light.
Among such methods, a thermal lens spectrometry of using a thermal lens effect is also known as a most sensitive detection method. When a laser beam is focused with a condenser lens and is incident on a measuring object, heat generates near its focus (focal point) by the above-described photothermal effect, and temperature at the point rises. Since the spatial intensity distribution of the laser beam in the above-described focus is generally a gaussian type, the heat distribution, generated in proportion to the intensity distribution, and the temperature distribution generated as its result also become gaussian types. Then, since a refractive index of a solvent decreases as temperature rises, the refractive index distribution becomes a reversal gaussian type. Since this refractive index distribution can be assumed to be equivalent to a concave lens optically, and such refractive index distribution is called a thermal lens. This thermal lens spectrometry has another excellent characteristic that this method has 100 times or more of sensitivity as high as that of the absorption photometry in addition to a characteristic that a measuring object should just absorb the light with wavelength for excitation similarly to the absorption photometry.
In such a thermal lens spectrometry, there are a single beam method of performing both the excitation and detection of a thermal lens with one laser, and a double beam method using two separate lasers for excitation and detection of a thermal lens. Although the single beam method is characterized in simple structure and easy optical adjustment, it becomes difficult to set the optimal optical configuration for each of excitation and detection since one laser performs both the excitation and the detection of a thermal lens, and sensitivity is low in comparison with the double beam method.
On the other hand, since the double beam method can use separate lasers for the excitation and the detection of a thermal lens, it is possible to set the optimal optical configuration for each, and to realize high sensitivity. Then, many examples of such a double beam method are known.
In addition, there is an example where highly sensitive measurement was performed with applying this double beam method to μ-TAS (Manabu Tokeshi et al., J. Lumin., Vol.83–84, 261–264, 1999). In this double beam method, an Ar ion laser is used as an excitation light source, and a helium neon laser is used as a detection light source (hereafter, detection light is described as probe light), after making these two laser beams coaxial, the beams are led to a microscope, and are focused with an objective lens on a sample in a groove engraved on a chip.
In such a conventional thermal lens spectrometry, a gas laser such as an Ar ion laser or a helium neon laser, a dye laser excited by a gas laser, or the like has been generally used. However, presently, when an apparatus generating the above-described laser beams is actually used, the apparatus is large-sized, large-scale cooling means such as water cooling means is needed at the time of a high-power output, and the apparatus becomes very expensive. In order to solve those problems, several examples that use semiconductor lasers and are comparatively small systems are known.
First, examples using the single beam method will be described. In Japanese Patent Laid-Open No. 4-369467, a semiconductor laser is used, further, in order to shorten distance between a sample and a detector, an optical system which detects a focus error of reflected light is adopted, and the miniaturization of an optical head is realized.
In addition, there is also an example where an apparatus which is small and portable is realized with the single beam method using the semiconductor laser with a wavelength of 670 nm, and further, an entire system is miniaturized by connecting a sample and a detector with a fiber (KIM S-H, Bull. Korean Chem. Soc., Vol. 18, 108–109, 1997, and KIM S -H et al., Bull. Korean Chem. Soc., Vol. 17, 536–538, 1996).
On the other hand, there is also an example using the double beam method. For example, phosphorus was analyzed by making a semiconductor laser with a wavelength of 824 nm be an excitation light source, and the detection limit of 0.35 ppb was obtained in an aqueous solution (K. Nakanishi et al., Anal. Chem., Vol. 57, 1219–1223, 1985).
FIG. 7 shows a structural diagram explaining the construction of a conventional photothermal spectroscopic analyzer using the double beam method which uses semiconductor lasers. In such a photothermal spectroscopic analyzer, excitation light is outputted from a semiconductor laser beam-emitting apparatus 71, and after being focused with a lens 72, the light is focused with a condenser lens 73 on a sample in a glass sample cell 75 with the optical path length of 1 cm. Then, a thermal lens is formed in the above-described sample where the above-described excitation light is incident.
In addition, probe light outputted from a helium neon laser apparatus 81 is led to the sample cell 75 by a beam splitter 74 in collimated light coaxially with the above-described excitation light. The probe light incident on the above-described thermal lens is given a thermal lens effect in the sample cell 75, reflected by a mirror 76, and focused by a condenser lens 77, and thereafter, the probe light is received by a photodiode 80 through an excitation light cut-off filter 78 and a pinhole 79, and is given a signal analysis.
Similarly, there is also an example where the detection limit of 8×10−5 M is obtained with using Nd3+ aqueous solution and a 10mW excitation light output by using a GaAlAs semiconductor laser with the wavelength of 795 nm as an excitation light source (D. Rojas et al., Rev. Sci. Instrum., Vol. 63, 2989–2993, 1992).
Furthermore, in order to improve sensitivity, there is also an example where the absorbance limit of 1.1×10−3 was obtained in an aqueous solution by increasing an output of excitation light to 100 mW by using an array type semiconductor laser with the wavelength of 818 nm (Cladera Forteza et al., Anal. Chem. Acta Vol. 282, 613–623, 1993).
However, each of these three examples uses a helium neon laser, which is comparatively large-sized and expensive, as probe light, and hence, this is not an apparatus composed of only semiconductor lasers.
As mentioned above, the photothermal spectrocopy method is highly sensitive in comparison with the absorption photometry which analyzes a sample by using the absorption of light similarly, and it is possible to miniaturize a photothermal spectroscopic analyzer to some extent by making a semiconductor laser be an excitation light source.
However, the above-described conventional technology has the following problems when realizing the photothermal spectroscopic analyzer which is equipped with high sensitivity, high accuracy, maintenance-free performance, short start-up time, and high reliability and operability in addition to the natural requirements for performing a POC analysis etc., that is, small dimensions for portable use and inexpensiveness.
First, as mentioned above, the single beam method using a semiconductor laser has an advantage that the adjustment of an optical system becomes easy. However, since its sensitivity is low in comparison with the double beam method, its sensitivity is insufficient in many cases as a method of using this in the case, where high accuracy is required in data, such as medical diagnosis or an environmental analysis.
Next, in the conventional double beam method, only an excitation light source is composed of a semiconductor laser, and a helium neon laser that is large-sized and expensive is still used as a probe light source. In such a case, it is reported that the minimum size of an optical system except the light sources is 30 cm×30 cm. However, since the size of the helium neon laser which is a light source is usually 5 cm dia.×20 cm long, the apparatus become large-sized when this is added (D. Rojas et al., Rev. Sci. Instrum., Vol. 63, 2989–2993, 1992).
In addition, since a large-sized laser such as a helium neon laser is used, a light source and an optical system cannot be integrated and the light source and optical system are separately fixed on an optical bench, and hence, carrying is impossible. Furthermore, a gas laser also has troubles such as necessity of a high voltage power supply.
Moreover, up to now, in order to obtain sufficient measurement sensitivity, it is necessary to make distance from a sample to a device corresponding to a pinhole be 1 m or more that is long. Thus, since long distance is necessary for leading the probe light, which is given the thermal lens effect in the sample, to a pinhole, the miniaturization of the whole optical system is disturbed due to the restriction of such distance from the sample to the device. If this distance is shortened without design, it is expected that it leads to sensitivity deterioration.
In addition, there is an example where the above-mentioned distance is shortened by not using the pinhole method as the light-receiving method but adopting a method using an optical system which directly detects a focus error. However, there is no report of affirming that the sensitivity of this method is superior to that of the pinhole method (Japanese Patent Laid-Open No. 4-369467 applied by YOKOGAWA ELECTRIC CORP.).
Furthermore, as a method of improving the sensitivity of a thermal lens spectrometry, it is commonly known that it is important to optimize the degree of focusing to the depth of a sample cell (namely, optical path length) and to adjust a focal point of probe light with shifting the focal point from a sample (Thierry Berthoud et al., Anal. Chem., Vol. 57, 1216–1219, 1985). However, since the optimal degree of focusing or the optimal focal point of probe light depend on a plurality of other parameters and it is not possible to theoretically analyze all of those parameters systematically, there is no report of the theoretical analysis of the optimum values at the time of raise the degree of focusing, up to now.
In particular, since the optical path length becomes short in μ-TAS, it is expected that it is necessary to raise the degree of focusing to some extent. In order to raise the degree of focusing, it is needed to make the numerical aperture of a condenser lens large. Since a focal length becomes short to several cm when the numerical aperture is increased, it is not possible to make the excitation light and probe light be coaxial due to a spatial limitation in the case that the degree of focusing and the focal point of the probe light are adjusted by using separate condenser lenses for the excitation light and the probe light like a conventional way.
Then, it becomes possible to use a condenser lens with large numerical aperture since the excitation light and probe light are focused after being made to be coaxial if the condenser lens of the excitation light and probe light are made to be common like another conventional technology (Manabu Tokeshi et al., J. Lumin., Vol.83–84, 261–264, 1999). However, there is no report about an adjusting method of a focal point of probe light at the time of sharing a condenser lens.
In particular, since it is known that the outgoing light of a semiconductor laser completely differs from a gas laser, a simple method of adjusting a focal point that is suitable to the characteristics of the semiconductor laser is needed.
First, the outgoing light of a semiconductor laser is divergent light, and its cross sectional geometry becomes elliptic. Furthermore, if the outgoing light is focused as it is, the astigmatism that a focal point changes according to a cross sectional direction of focused light exists. Therefore, when using semiconductor lasers as both the light sources of the excitation light and probe light, it is necessary to correct the intrinsic characteristics of the outgoing light of these semiconductor lasers.
Thus, in the case of using semiconductor lasers as both the light sources of the excitation light and probe light, it is necessary to perform the above-described correction and to provide simple and inexpensive means for adjusting a focal point of the probe light.
In addition, when the numerical aperture of a condenser lens is made large and the degree of focusing is raised, it is necessary to accurately adjust the focal points of the excitation light and probe light in a sample cell. Up to now, since the positional relation of a focal point and a sample cell is visually adjusted by using a microscope, not only a visual alignment error arises, but also an alignment error by a measuring operator is included. In addition, it is impossible to automatically adjust an above-described positional relation with a machine in such visual method. Furthermore, an apparatus becomes large by using a microscope.
In addition, since not only about 10 minutes of time was necessary until being stabilized after switching on a power supply but also mechanical modulation means such as a chopper was required for a gas laser at the time of modulating an output, it was not so easy to perform miniaturization and cost reduction.
Furthermore, in addition to these, it is required that a photothermal spectroscopic analyzer used for POC analysis etc. should be strong in an environmental temperature change and vibration. Furthermore, it is desirable that the photothermal spectroscopic analyzer used for POC analysis etc. does not need a high voltage power supply and is able to be driven by a dry cell etc.
As described above, in conventional technologies, there is no measure for the characteristics naturally required of the photothermal spectroscopic analyzer used for POC analysis etc.
Then, a task of the present invention is to provide a photothermal spectroscopic analyzer which is equipped with all the requirements as an apparatus for POC analysis etc., such as small size, inexpensiveness, high sensitivity, high accuracy, maintenance-free performance, short start-up time, and possibility of automatic measurement by solving the problems which the above conventional photothermal spectroscopic analyzers have.